Use of nicotinic analogs for treatment of degenerative diseases of the nervous system

ABSTRACT

Method of using anabaseine, and DMAB-anabaseine for stimulating brain cholinergic transmission and a method of making anabaseine.

BACKGROUND OF THE INVENTION

This application is a 371 of PCT/US92/01451 filed Feb. 27, 1992.

FIELD OF THE INVENTION

This invention relates to anabaseine, DMAB-anabaseine, and anabasine andtheir use to treat degenerative diseases of the nervous system.

DESCRIPTION OF THE BACKGROUND ART

It has long been customary in classifying diseases of the nervous systemto group them as degenerative, thereby indicating they are characterizedby a gradually evolving, relentlessly progressive, neuronal death.Science has shown that a considerable portion of disorders that areclassed as degenerative are associated with genetic predisposition whichresults in a pattern of dominant or recessive inheritance. However,others, although they do not differ in a fundamental way from thehereditary disorders, may occur only sporadically as isolated instanceswithin a given family.

As a consequence, since by definition, classification of degenerativediseases cannot be based upon exact knowledge of their cause orpathogenesis, subdivision of these diseases into individual syndromesrests upon descriptive criteria based largely upon pathologic anatomyand consideration of clinical aspects. As a result, this group ofdiseases presents itself in the form of several clinical syndromes.However, apart from the general differences that allows the distinctionof one syndrome from another, there are certain general attributes whichtypify this entire class of disorders.

The degenerative diseases of the nervous system can typically be dividedinto disorders characterized by progressive dementia in the absence ofother prominent neurologic signs (e.g., Alzheimer's disease, seniledementia, and Pick's disease); syndromes which combine progressivedementia with other prominent neurologic abnormalities (e.g.,Huntington's disease, Hallervorden-Spatz, and progressive familialmyoclonic epilepsy); syndromes of gradually developing abnormalities ofposture and movement (e.g., Parkinson's disease, striatonigraldegeneration, torsion dystonia, and Gilles de la Tourette syndrome);syndromes of progressive ataxia (e.g., cerebellar cortical degeneration,olivopontocerebellar atrophy, and Friedgreich's ataxia); and syndromesof muscular weakness and wasting without motor neuron disease (e.g.,amyotrophic lateral sclerosis, spinal muscular atrophy, and hereditaryspastic paraplegia), to name but a few.

Among those diseases listed above, perhaps those most familiar areAlzheimer's and Parkinson's diseases. These diseases are progressiveneurological disorders characteristically associated with aging.Alzheimer's disease is characterized by a profound loss of memory andother cognitive functions, while Parkinson's disease is anextrapyramidal movement disorder. Both are invariably fatal. Althoughthere is no effective treatment for Alzheimer's disease, clinical trialsare underway with several drugs that increase brain cholinergictransmission. In Parkinson's disease, several treatments are temporarilyuseful, notably L-DOPA related therapies that replace dopamine in thenigrostriatal pathway. However, in Parkinson's disease the therapeuticefficacy of even the best drugs is temporary at best.

Although the loss of neurons in the late stages of Alzheimer's diseaseis profound, only a few neuronal pathways appear to be affected in itsearliest stages. These include cholinergic projections from the nucleusbasalis to the cerebral cortex and from the septum to the hippocampus,noradrenergic projections from the locus cerululus to the cerebralcortex, and several peptidergic neurons that are probably intrinsic tothe cerebral cortex. The loss of the aforementioned cholinergic pathwaysin particular is believed to underlie the early memory loss, since thesepathways are known to be important for memory and cognition. Thisassociation accounts for the major emphasis in novel cholinergictreatments for Alzheimer's disease, at least in its early stages.

A recent study on Alzheimer's disease demonstrated that loss ofcholinergic projections from the nucleus basalis to the cerebral cortexwas sufficient, after extended intervals, to cause trans-synaptic neuronloss in the rat. Thus, it is conceivable that the early loss ofanalogous cholinergic neurons in Alzheimer's disease could cause aprofound cascade phenomenon resulting in the loss of many neurons over aperiod of years. If so, then replacement therapy might not only improvesurvival of these neurons, but perhaps more important, keep other braincells from dying.

Given the possibility of such therapy, it is of primary importance todetermine the type of cholinergic agent most likely to improve memoryand/or keep brain neurons from dying after the loss of cholinergicneurons. To address this issue, it is necessary to consider the twogeneral types of cholinergic transmission in the brain. One is termedmuscarinic, the other nicotinic. These terms are based on the type ofreceptor to which acetylcholine binds to in order to elicit itsneurotransmitter effect. In brain regions associated with memory, themuscarinic receptors predominate quantitatively over the nicotinicreceptors, although both types coexist. For this reason, mostinvestigators traditionally focused on the development of muscarinicagonists to improve memory-related behaviors. These agents have beenfound to have moderate effects in rats with lesions of the nucleusbasalis, but have little effect in patients with pronounced Alzheimer'sdisease.

There is reason to believe, however, that nicotinic transmission mayalso be important for treating Alzheimer's disease. This is supported bythe fact that cerebral cortical nicotinic receptors decreasesignificantly during the disease, while post-synaptic muscarinicreceptor levels are often unchanged. These observations are consistentwith the hypothesis that neurons expressing nicotinic receptors are lostin the disease. When these observations are combined with those of thepresent inventors, that lesions of ascending cholinergic neurons fromthe nucleus basalis cause a trans-synaptic neuron loss in the cortex, itis hypothesized that the neurons in the cortex that dietrans-synaptically (and in Alzheimer's disease) do so because they donot receive enough nicotinic stimulation. For this reason, the inventorsbelieve nicotinic agents may be useful as replacement therapy forkeeping brain neurons alive in Alzheimer's disease that would otherwisedie from lack of nicotinic transmission. An analogous situation existsin several other systems such as: (a) muscle cells, which atrophy in theabsence of nicotinic activation; (b) sympathetic ganglia, which requireeither nerve growth factor or nicotinic transmission (in the presence ofcalcium ions) in order to survive in culture; and (c) nigrostriataldopamine neurons, which appear to be partially spared by nicotinefollowing lesions of the substantial nigra. Also, it is important tonote that there exist several types of nicotinic receptors in the brain,which allows considerable potential selectivity in targeting drugs forcertain nicotinic sites.

The observation that nicotine treatment can preserve nigrostriataldopamine neurons in an animal model for Parkinson's disease isconsistent with epidemiological evidence that there is a lower incidenceof this disease in cigarette smokers (even after adjusting for thesmoking-induced increase in mortality). The mechanism whereby nicotinecan preserve these neurons is not known, but it does appear to involveeffects of nicotinic transmission on dopamine neurons themselves, sincethese neurons possess this type of cholinergic receptor. While theremainder of this patent application focuses on the potential treatmentof Alzheimer's disease with nicotinic receptor agents, it should benoted that these drugs may be just as effective, or more so, ondopaminergic neurons that are lost in Parkinson's disease.

Nicotine has been used in several clinical trials for the treatment ofAlzheimer's disease, primarily over rather short intervals for itspotential memory enhancing effect (not for its ability to block longterm trans-synaptic cell loss). In one recent study, nicotine had amarginally positive effect on memory and an even greater one ofimproving the mood of the patients. These positive results have not beenfollowed up with longer term ones, however. Unfortunately, whilenicotine has a history of improving memory related behaviors in humansand animals, its potent toxicity, low effective dose range, andperipheral side effects, have basically rendered it unacceptable fortreating Alzheimer's disease.

Thus, considerable need exists for agents which stimulate cholinergictransmission, but, unlike nicotine, are relatively non-toxic. Thepresent invention provides a method of using agents which have thiscapability.

SUMMARY OF THE INVENTION

The present invention arose out of the discovery that anabaseine,DMAB-anabaseine, and anabasine could be used to improve overall brainneurocortical cholinergic activity. The interaction of these agents withnicotinic receptors has decreased levels of toxicity as compared tonicotine.

In the absence of long term studies for the clinical effectiveness ofnicotine or its analogs for degenerative neural diseases, such asAlzheimer's or Parkinson's disease, the present invention has developedthe nucleus basalis lesioned rat as a model for trans-synaptic neuronaldegeneration caused by the loss of ascending neurons. Bilateral lesionsof cholinergic neurons in the nucleus basalis were induced with ibotenicacid to cause long-term, essentially irreversible deficits inneocortical choline acetyltransferase activity, an enzyme selective forcholinergic neurons. However, passive avoidance behavior, a learning andmemory paradigm particularly sensitive to nucleus basalis-lesions,reportedly recovers to normal levels sometime between 2-8 monthspost-lesioning.

Various other aspects and attendant advantages of the present inventionwill be more fully appreciated from an understanding of the followingdetailed description in combination with the accompanying examples.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Effects of NBM lesions on anabaseine-induced improvement inpassive avoidance behavior.

FIG. 2 Effects of NBM lesions on DMAB-anabaseine-induced improvement inpassive avoidance behavior.

FIG. 3 Effects of anabasine on passive avoidance behavior.

FIG. 4 Effect of DMAB-anabaseine on performance of aged rats in the17-arm radial maze.

FIG. 5 Effect of DMAB-anabaseine on performance of aged rats in theLashley III maze.

FIG. 6 Aspartate release from brain tissue exposed to anabaseine.

FIG. 7 Aspartate release from brain tissue exposed to DMAB-anabaseine.

DETAILED DESCRIPTION OF THE INVENTION

Anabaseine, 2-(3-pyridyl)-3,4,5,6- tetrahydropyridine, occurs in certainmarine worms, which use the substance to paralyze prey and deterpredators (Kem, et al., Toxicon, 9:23, 1971). Anabaseine is a potentactivator of vertebrate neuromuscular nicotinic acetylcholine receptors(Kem, Amer. Zoologist, 25:99, 1985). Both nicotine and anabaseinepossess a non-aromatic ring attached to the 3-position of a pyridylring. Anabaseine's non-aromatic tetrahydropyridine ring imine doublebond is conjugated with π-electrons of the 3-pyridyl ring. The iminenitrogen is a much weaker base than the pyrrolidinyl nitrogen ofnicotine (Yamamoto, et al., Agr. Biol. Chem., 26:709, 1962).Considerable evidence (Barlow and Hamilton, Brit. J. Pharmacol., 18:543,1962) exists that the non-aromatic ring nitrogen of nicotine must beprotonated (cationic) in order to avidly bind to the skeletal musclenicotinic receptor and activate the opening of its channel. Atphysiological pH, anabaseine also exists in a hydrolyzed ammonium-ketoneform as well as the cyclic imine (unionized) and cyclic iminium(monocationic) forms. The inventors have determined that anabaseine actsas a central nicotinic receptor agonist primarily through its cycliciminium form.

The synthesis of anabaseine was first reported in 1936 (Spath, et al.,Chem. Ber., 69:1082, 1936). Unfortunately, this technique utilized anelaborate isolation scheme involving a distillation and preparation of apicrate. Medicinally, the picrate is of no useful value, in fact, sincepicrate is toxic and potentially explosive, its presence precludes thedirect use of anabaseine in physiological systems when produced by thistechnique.

The first analog of anabaseine to be synthesized was3-[p-(dimethylamino) benzylidene]-3,4,5,6-tetrahydro-2,3'-bipyridine,also termed DMAB-anabaseine (Kem, et al., Toxicon, 9:23, 1971). Thiscompound, resulting from the electrophilic attack of Ehrlich's reagentupon anabaseine, is a stable orange-colored compound.

The present invention provides an improved method for synthesizinganabaseine which overcomes the problems associated with prior disclosedtechniques for its synthesis.

The first part of the improved synthesis of anabaseine, the joining ofan activated derivative of nicotinic acid and a modified 2-piperidone,is performed using a mixed Claisen condensation. The second part of thesynthesis involves the hydrolysis and decarboxylation of the condensedproduct. The overall reaction sequence is shown below. ##STR1##

In the scheme presented herein, certain protecting and activating groupsare specifically illustrated. However, one skilled in the art willrecognize that other protecting and activating groups could have beenused. For example, a variety of amino protecting group can be used toprotect the nitrogen of 2-piperidone (1). Representative aminoprotecting groups are C₁ -C₄ alkanoyl, benzyl, and trialkylsilylderivatives such as trimethylsilyl and butyldimethylsilyl. The preferredamino protecting group is trimethylsilyl (TMS). The TMS-protected2-piperidone (2) is prepared by deprotonation and subsequent reactionwith trimethylchlorosilane. Typical silylation conditions are the use oflithium diisopropylamide (LDA) in an inert solvent such astetrahedrofuran (THF) at -70° C. For each one mole of 2-piperidone, atleast one mole of LDA, preferably 11/2 moles, should be used to ensurecomplete silylation. While maintaining the temperature at -70° C., atleast one molar equivalent of TMS is combined with the LDA-addedreaction mixture. Normally, silylation is complete within a few hours byraising the reaction temperature to ambient temperature.

The protected 2-piperidone (2) is next enolyzed to an enolate by base.Conveniently, this enolization can be conducted by simply addingadditional LDA to the reaction mixture containing compound (2). Althoughthis is a preferred process, other suitable bases which can be employedinclude metal amides such as NaNH₂ or KNH₂, metal hydrides such as NaHor KH, and metals such as Na or K. In practice, the reaction mixture iscooled to -70° C., at which point at least one molar equivalent of LDAis added. Enolization is usually complete within an hour, and theresultant amide enolate (3) can be directly used in the nextcondensation reaction.

The key Claisen condensation between a 2-piperidone enolate and anicotinic acid derivative can be carried out, e.g., by combining thelithium amide enolate (3) in an inert solvent such as THF with about onemolar equivalent of ethyl nicotinate. Reaction temperature can bevaried, but it is preferred to start the condensation at -70° C. and toallow the temperature to warm up to ambient temperature. Reactionrequires a few hours to 24 hours until its completion.

Although an ethyl ester form of nicotinic acid (4) has been illustratedhereinabove, activation of the carboxylic group to expedite condensationcan be achieved by other activating groups known in the art. Especiallyuseful in the herein described condensation are anhydrides, particularlycyclic anhydrides, acid halides, and activated esters such as thosederived from N-hydroxysuccimide and N-hydroxypthalimide. Alkyl esters ofup to C₅ other than ethyl ester can also be used.

The condensed product (5) is isolated after removal of TMS group byhydrolysis. The product (5) is normally subjected to hydrolysis anddecarboxylation without further purification.

Conversion of compound (5) to the final anabaseine (6) is accomplishedby first hydrolyzing compound (5) with a strong acid such asconcentrated hydrochloric acid; and by second decarboxylating theintermediate β-keto acid (not shown in the above scheme). Bothhydrolysis and decarboxylation steps are conveniently conducted inone-pot in the presence of concentrated hydrochloric acid at an elevatedtemperature, e.g., under reflux. Anabaseine (6) is thus obtained as itsdihydrochloride.

Anabasine is commercially available from Aldrich Chemical Co.Alternative sources of anabasine are reduction of anabaseine.

Reduction of anabaseine to anabasine can be achieved by several ways:(1) Hydrogeneration with hydrogen over Pd/C, as described in E. Spath etal., Chem. Ber. 69, 1082 (1936); (2) Borohydride reduction with eitherNaBH₃ CN or with NaBh₄, as described in E. Leate, J. Org. Chem. 44, 165(1979); and (3) Reduction with hot formic acid.

Anabasine having the following formula contains an asymmetric center atthe 2-carbon of the piperidine ring. ##STR2##

Thus, anabasine can exist as an optically active form. The presentinvention embraces such optically pure anabasine, the pure enantiomersthereof, and the racemate thereof.

Anabaseine and anabasine in their free base form will form acid additionsalts, and these acid addition salts are non-toxic and pharmaceuticallyacceptable for therapeutic use. The acid addition salts are prepared bystandard methods, for example by combining a solution of anabaseine oranabasine (base) in a suitable solvent (e.g., water, ethyl acetate,acetone, methanol, ethanol or butanol) with a solution containing astoichiometric equivalent of the appropriate acid. If the saltprecipitates, it is recovered by filtration. Alternatively, it can berecovered by evaporation of the solvent or, in the case of aqueoussolutions, by dyophilization. Of particular value are the sulfate,hydrochloride, hydrobromide, nitrate, phosphate, citrate, tartrate,pamoate, perchlorate, sulfosalicylate, benzene sulfonate, 4-toluenesulfonate and 2-naphthalene sulfonate slats. These acid addition saltsare considered to be within the scope and purview of this invention.

As a result of using the above method for the synthesis of anabaseine:(1) the chemistry is cleaner and simpler; (2) higher yields ofanabaseine are obtained; and (3) picric acid is not present, such that amore directly pharmacologically useful form of anabaseine is produced.

The term "therapeutically effective" means that the amount of nicotinicreceptor agent used is of sufficient quantity to increase braincholinergic transmission. The dosage ranges for the administration ofthe agent of the invention are those large enough to produce the desiredeffect in which the nicotinic receptors show some degree of stimulation.The dosage should not be so large as to cause adverse side effects, suchas unwanted cross-reactions, anaphylactic reactions, and the like.Generally, the dosage will vary with the age, condition, sex, and extentof the disease in the patient and can be determined by one of skill inthe art. The dosage can be adjusted by the individual physician in theevent of any contraindications. Dosage can vary from about 1 μg/kg/doseto about 1000 μg/kg/dose, preferably from about 10 μg/kg/dose to about500 μg/kg/dose, most preferably from about 30 μg/kg/dose to about 100μg/kg/dose in one or more dose administrations daily, for one or severaldays. Alternatively, the dosage can be administered indefinitely inorder to prevent a recurrence of cognitive function loss, for example,by administration of the agent in a slow-release form.

The nicotinic receptor agent of the invention can be administeredenterally, parenterally, or by gradual perfusion over time. Thenicotinic receptor agent of the invention can be administeredintravenously, intraperitoneally, intramuscularly, subcutaneously,intracavity, transdermally, or orally.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose, and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like. In order to form a pharmaceutically acceptablecomposition suitable for effective administration, such compositionswill contain an effective amount of the nicotinic receptor agent,together with a suitable amount of a carrier vehicle.

Additional pharmaceutical methods may be employed to control theduration of action. Controlled release preparations may be achieved bythe use of polymers to complex or adsorb the nicotinic receptor agent.The controlled delivery may be exercised by selecting appropriatemacromolecules (for example, polyesters, polyamino acids, polyvinylpyrrolidone, ethylenevinylacetate, methylcellulose,carboxymethylcellulose, and protamine sulfate) and the concentration ofmacromolecules as well as the methods of incorporation in order tocontrol release. Another possible method to control the duration ofaction by controlled release preparations is to incorporate thenicotinic receptor agent into particles of a polymeric material such aspolyesters, polyamino acids, hydrogels, poly (lactic acid) or ethylenevinylacetate copolymers. Alternatively, instead of incorporating thenicotinic receptor agent into these polymeric particles, it is possibleto entrap the nicotinic receptor agent in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization,for example, hydroxymethylcellulose or gelatin-microcapsules and poly(methylmethacrylate) microcapsules, respectively, or in colloidal drugdelivery systems, for example, liposomes, albumin microspheres,microemulsions, nanoparticles, and nanocapsules or in macroemulsions.Such teachings are disclosed in Remington's Pharmaceutical Sciences(17th Ed., A. Oslo, ed., Mack, Easton, Pa. 1985).

The invention also relates to a method for preparing a medicament orpharmaceutical composition comprising the nicotinic receptor agent ofthe invention, the medicament being used for therapy to stimulate braincholinergic transmission.

The above disclosure generally describes the present invention. A morecomplete understanding can be obtained by reference to the followingspecific examples which are provided herein for purposes of illustrationonly, and are not intended to limit the scope of the invention.

EXAMPLE 1 SYNTHESIS OF ANABASEINE

A dihydrochloride crystalline form of anabaseine was prepared via theinitial synthesis of 3-nicotinoyl-2- piperidine enolate which was thenhydrolyzed and decarboxylated to yield anabaseine.

1) Preparation of 3-Nicotinoyl-2-piperidine Enolate ##STR3## a) Reaction

A 250 mL flask equipped with a nitrogen inlet was flame dried andcharged with nitrogen. Dry THF (40 mL) was added to this flask andcooled to -70° C. in a dry ice/acetone bath before 38 mL (57 mmol, 1.5eq) of 1.5M LDA in cyclohexanes (from Aldrich) was added. A solution of5.68 g (57.3 mmol, 1.5 eq) of 2-piperidone (previously dried) in 15-20mL of THF (distilled from sodium and benzophenone to dry) was addedthrough a cannula over a period of 20 min to the stirring LDA solutionat -70° C. to form the deprotonated amide. While stirring the reactionmixture at -70° C., 7.2 mL (56.7 mmol, 1.5 eq) of trimethylsilylchloride was added through an oven-dried syringe all at once. Theresulting solution was stirred at -70° C. for 15 min and at roomtemperature or 2 hrs to form the TMS protected piperidone. The solutionturned milky colored and a solid precipitate (thought to be LiCl) formedafter a few minutes at -70° C. The precipitate dissolved and thesolution was clear yellow at room temperature. The reaction mixture wascooled back down to -70° C., before another 38 mL (57 mmol, 1.5 eq) of1.5M LDA was added with stirring to form the amide enolate. Afterstirring the reaction mixture at -70° C. for 20 min, 5.2 mL (38 mmol, 1eq) of ethyl nicotinate was added. The reaction mixture was stirredvigorously at -70° C. for 20 min and at room temperature for 17 hrs.After stirring at room temperature for 30 min the reaction mixture wascloudy and after 90 min the reaction mixture contained a precipitate.The yield can be increased if 2 equivalents of the protected2-piperidone enolate are used instead of 1.5 equivalents.

After stirring for 17 hrs at room temperature, the reaction mixture wasthick with cream colored precipitate (product). Water (50 mL) was addedand the reaction mixture was stirred for 15 min to hydrolyze the TMSprotecting group. The thick pasty precipitate was filtered out of thereaction mixture. The precipitate appeared to pick up water on standingbut then formed a stable pale yellow solid. The solid was dried in adrying pistol to yield 8.060 g of pale yellow powdery solid product(mp>250° C.). This solid was used without further purification.

The remaining phases (water and organic) from the reaction mixture canbe checked for product using ferric nitrate (Fe(NO₃)₃). An aqueoussolution of ferric nitrate turns dark blue or purple in the presence ofa compound. Add a couple drops of ferric nitrate solution to aneutralized sample of the aqueous or organic phases from the reactionmixture to check for additional product.

2) Hydrolysis/Decarboxylation of 3-Nicotinoyl-2-piperidone Enolate toAnabaseine ##STR4## a) Reaction

The lithium enolate of 3-nicotinoyl-2-piperidone (4.94 g) of step 1 wasadded slowly to a round bottom flask containing 30 mL of concentratedHCl which was chilled in an ice bath and stirred. The enolate was notreadily soluble. The reaction mixture was heated at reflux undernitrogen overnight to effect the hydrolysis and decarboxylation. Theproduct, anabaseine dihydrochloride, was very water soluble. Thereaction should not be diluted with too much aqueous acid or the productwill not crystallize during the work up.

Next, the reaction mixture was cooled to room temperature and dilutedslowly with isopropyl alcohol to a volume of about 350 mL. The isopropylalcohol solution was cooled in the refrigerator and the product slowlycrystallized. The solution was allowed to warm to room temperaturebefore filtering the 3.88 g of white needle-like crystalline solid (mp173°-178° C., decomp). The filtrate was cooled in the refrigerator toyield 0.209 g of a second crop of product.

The first crop of solid was recrystallized by adding it to about 200 mLof hot isopropyl alcohol and adding 6M HCl slowly to the boiling mixtureuntil all of the solid dissolved (about 5 mL of HCl was added). Aftercooling the solution in the refrigerator, 3.26 g of anabaseinedihydrochloride was collected (mp 175°-180° C., decomp). Anabaseinedihydrochloride was prepared in 56% overall yield based on the moles ofethyl nicotinate used

Since the dry crystalline solid product is not hygroscopic, but the wetsolid may pick up water after filtration, filtration should beperformed, for example, under nitrogen atmosphere.

EXAMPLE 2 EFFECT OF ANABASEINE, DMAB-ANABASEINE AND ANABASINE ONMEMORY-RELATED BEHAVIOR

A. Passive Avoidance Behavior

Male Sprague Dawley albino rats were used for all studies and weremaintained in departmental animal facilities, using NIH guidelines forcare of animals. Where lesioned animals were tested, lesions wereinduced in anesthetized animals by bilateral infusion of ibotenic acid(5 μg in 1 μl) or phosphate buffered saline (PBS) into the nucleusbasalis region.

For passive avoidance behavior, animals received a moderately strongshock (0.8 m Amp) for 1 second after entering a dark room. After 24hours, the animals were again tested to determine if they could rememberto stay out of the dark room. Animals were only allowed 5 minutes tomake their choice, when they were removed from the lighted chamber. Fortesting the effects of drugs in animals that were not lesioned, shockswere only 0.5 m Amp in intensity, and in the animals were allowed 72hours until they were tested after training. In all drug-treatmentstudies, the drugs were injected intraperitoneally in saline diluent 5minutes before the trial and 5 minutes before the testing period.

As shown in FIGS. 1 and 2, anabaseine and DMAB-anabaseine, respectively,were more potent in lesioned then in unlesioned animals. For nicotine,0.05 mg/kg was effective in unlesioned animals, while 0.02 mg/kg waseffective in lesioned animals. For anabaseine, a similar 2.5 fold shiftwas observed. For both of these drugs, the animals were also moresensitive after lesioning in that 0.2 mg/kg doses interfered withtraining or behavior. For DMAB-anabaseine, potency was increased between2-2.5 fold by lesioning.

The effect of (-)anabasine on passive avoidance was also determinedusing unlesioned animals (FIG. 3). In these experiments (-)anabasine wasinjected intraperitoneally 5 min. before training and testing in thepassive avoidance apparatus. Only animals that trained within 300 secthe first time were used (i.e., those animals that entered the darkcompartment and received a mild foot shock).

These results indicate that anabaseine, DMAB-anabaseine, and anabasinecan improve this type of memory-related behavior, apparently by bindingto and activating nicotine transmission, even in animals with reducedneocortical cholinergic activity. This latter state mimics that seen inAlzheimer's disease.

B. Radial Maze Testing

The 17 arm radial maze requires animals to remember a baited set of 8arms out of the 17 total arms. At the start of each daily trial, ratsare placed in the center of the maze and permitted to choose among the17 arms until all 8 food rewards are taken or until fifteen minuteselapse. Those animals that reach a performance criteria of 17 armchoices on two consecutive days of testing during the first 14 days oftesting are continued in testing for an additional 30 days. For suchanimals, only data collected after day 14 are used in the statisticalanalyses. Statistical analyses are done on 3 sets of data. The first isa measure of general learning: the percent correct choices (entries intobaited arms) over the first 8 arm choices. The second is a measure ofshort term memory (working memory) calculated from the first 12 armchoices: the percent of choices into baited arms (containing food) overthe total number of choices within the baited set. Working memory, aninteratrial measure of short term memory, measured the rat's ability toremember which of the arms in the baited set were previously entered andthe food reward taken. The third set of data measured long-term orreference memory and also was calculated from the first 12 arm choices.Reference memory, defined as an inter-trial measure, is the percentcorrect choices in the baited set over the total number of arm choices.

Two groups of aged rats were tested in the 17-arm radial maze. One groupas given 0.2 mg/kg nicotine (n=5) and the other 2 mg/kg DMAB-anabaseine(n=5) at 15 minutes prior to each daily trial. The purpose of theseinjections was to determine if activation of nicotinic receptors (bynicotine or DMAB-anabaseine) can enhance the poor learning ability andlong term memory of aged rats in this task.

As shown in FIG. 4, DMAB improved a measure of long term memory withoutaffecting the short term memory of the animals. This selective effect issometimes typical of nicotine and other memory/learning paradigms.

C. Lashley III Maze Testing

The Lashley III maze tests an animal's ability to learn a series ofleft-right alternation turns. Six alternation errors are possible forany given daily trial; chance performance level is 3 errors per trial.Previous studies have shown that young adult sham operated animalsquickly learn to reduce their number of alternation errors to near zeroby the end of the test period. By contrast, 23 months old (Aged) shamoperated animals made substantially more errors over the 25 days oftesting. Moreover, bilateral nucleus basalis lesioning of aged ratsresulted in an even greater learning deficit compared to aged,sham-lesioned animals. Therefore, both age- and lesion-induced learningdeficits were observed. Nonetheless, all groups did improve theirperformance over time.

Aged animals injected with saline or DMAB-anabaseine were evaluated inthe Lashley III maze (FIG. 5). When injected at a 2 mg/kg dose beforetraining, DMAB reduced the number of errors that the aged animals madein this maze over the first two blocks of tests. This reflected animprovement in another memory-related behavior with this nicotineagonist.

EXAMPLE 3 EFFECT OF ANABASEINE AND DMAB-ANABASEINE ON NEUROTRANSMITTERRELEASE

Neurotransmitter release from synaptosomes provides a potential markerfor receptor-activity at different types of nerve terminals.Neurotransmitter release from intact slices or minces provides a markerfor receptor activity at many sites on the neuron. A comparison of theeffects of nicotine and other drugs on synaptosomes versus slicesprovides some idea as to the location of nicotinic receptors ondifferent types of cerebral cortical neurons, as well as their cellularlocation.

These different types of cerebral cortical transmitter systems have beentested. The first is the cholinergic; the procedures used to loadcholinergic neurons or slices with newly synthesized [3H]ACh weredescribed previously (Meyer, et al., 1987). The second is aspartate, anexcitatory amino acid which, like glutamate, is associated with memory(long term potentiation) and neuropathology (e.g., stroke). The thirdtype of neurotransmitter is GABA, which is the predominant transmitterin the cerebral cortex and is therefore very likely to receivecholinergic innervation.

In order to measure aspartate or GABA release, tissues were incubatedwith 100 nM [3H] aspartate or 250 nM [3h] GABA in Krebs Ringer buffer at37° C. for 30 minutes, then washed them in ice cold buffer. Allrelease-incubations were at 37° C. for 15 minutes in the presence orabsence of 50 mM KCl (depolarization). Radiation accumulation was alsomeasured in slices in order to express the released levels oftransmitter as % of total transmitter, since slices were somewhatvariable with respect to accumulation of label. K+ induced release oftransmitter was determined by subtracting the basal release from that inthe presence of the elevated K+, such that only the incremental releasewas determined.

Neurotransmitter levels (aspartate, glutamate) and enzyme levels wereassayed as described by Arendash, et al., Science, 238:952, 1987.Nicotine, anabaseine, and DMAB-anabaseine were found to have no effecton the basal or 50 mM KCl induced release of newly synthesized [3H]AChform synaptosomes. Also, no effect was seen on the release of [3H]aspartate from isolated terminals. Consequently, there do not appear tobe nicotine receptors or aspartate terminals (or glutamate terminals,since aspartate may be taken and released from glutamate terminals) inthe cerebral cortex.

In studies on brain tissue slices, nicotine (100 nM) increased the K+induced release of aspartate from slices without affecting thespontaneous release of transmitter (FIG. 6). The fact that nicotine candirectly depolarize aspartate neurons without increasing basal releaseas well as K+ induced release is surprising. One hypothesis is thatnicotine stimulates another type of neuron (presumably excitatory) todis-inhibit the release of aspartate; this dis-inhibition would not beseen except when the aspartate neuron itself was activated bydepolarization.

Interestingly, anabaseine and DMAB-anabaseine (except at one lowconcentration) did not increase aspartate release in a dose-relatedmanner (FIGS. 6 and 7). Thus, the effect on depolarization-inducedaspartate release was correlated with inhibition of high affinity [3H]nicotine binding, not the inhibition of [3H]ACh or [3H]methcarbacholbinding

This pattern was also observed with [3H]ACh release and [3H]GABA releasefrom cortical slices. Nicotine (100 nM) increased the K+ induced releaseof ACh but reduced the K+ induced GABA release from slices, whileanabaseine (1 μM) had no effect on either process. Nicotine alsoincreased basal ACh release, suggesting a direct excitatory effect onintrinsic cholinergic cell bodies (not terminals, from synaptosomestudies described above). Thus, it appears that the ability of nicotinictypes of compounds to modulate neurotransmitter release is not mediatedthrough one of the receptors with high affinity for anabaseine, orDMAB-anabaseine.

The invention now being fully described, it will be apparent to one ofordinary skill in the art that many changes and modifications can bemade without departing from the spirit or scope of the invention.

We claim:
 1. A method of treating degenerative memory dysfunction in ananimal comprising administering to the animal a therapeuticallyeffective amount of a nicotinic receptor agent, wherein the agent isselected from the group consisting of anabaseine, and DMAB-anabaseine.2. The method of claim 1, wherein the administration is parenteral. 3.The method of claim 2, wherein the parenteral administration is bysubcutaneous, intramuscular, intraperitoneal, intracavity, transdermal,or intravenous injection.
 4. The method of claim 1, wherein theadministration is enteral.
 5. The method of claim 1, wherein theadministration is at a dosage of about 1 μg/kg/dose to about 1000μg/kg/dose.
 6. The method of claim 1, wherein the administration is at adosage of about 10 μg/kg/dose to about 500 μg/kg/dose.
 7. The method ofclaim 1, wherein the administration is at a dosage of about 30μg/kg/dose to about 100 μg/kg/dose.
 8. The method of claim 1, whereinthe animal is human.
 9. The method of claim 1, wherein the memorydysfunction is dementia.